Thin film particle sensor

ABSTRACT

A Coulter-style, microfluidic sensor formed by stacking a plurality of substantially non-electrically conductive layers, typically formed from thin polymer films. Certain layers carry patterned electrodes that are arranged to permit their connection to an electrical interrogation circuit. Electrodes may be disposed in a 3-dimensional array in the sensor. A fluid path through the sensor includes an orifice sized to promote single-file travel of particles. The orifice may be defined by a tunnel passing through at least one layer. Particles entrained in an electrolytic carrier fluid may be detected, or otherwise characterized, by interrogation circuitry connected to the sensor. Certain sensors may include portions of a fluid path disposed parallel to the layers. In certain preferred embodiments, the sensor is carried by a cartridge, which is adapted to couple with an interrogation platform. Desirably, such coupling places the sensor in-circuit with operable interrogation electronics, as well as with a fluid-flow control device. Structure included in a cartridge may provide fluid sample loading, routing, and storage capabilities.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. utility applicationSer. No. 11/800,167, filed May 5, 2007 and claims the benefit under 35U.S.C. 119(e) of the filing date of Provisional Application Ser. No.60/798,155, filed May 5, 2006, for “Thin film particle sensor”, theentire disclosures of which are incorporated by reference.

BACKGROUND

1. Field of the Invention

This invention relates generally to electrically-based sensors for usein detecting, quantifying, qualifying, or otherwise sensing, particlescarried by a fluid. It is particularly directed to an improvedmicrofluidic sensor and interrogation structure for such particlesensing application.

2. State of the Art

Pioneering work in particle detection by measuring impedance deviationcaused by particles flowing through a small aperture between twocontainers of electrically conductive fluid is disclosed in U.S. Pat.No. 2,656,508 to W. H, Coulter. Coulter's name is now associated withthe principle of particles causing a change in electric impedance asthey occlude a portion of the aperture. Since publication of his patent,considerable effort has been devoted to developing and refining sensingdevices operating under the Coulter principle. Relevant U.S. patentsinclude U.S. Pat. Nos. 5,376,878 to Fisher, 6,703,819 to Gascoyne etal., 6,437,551 to Krulevitch et al., 6,426,615 to Mehta, 6,169,394 toFrazier et al., 6,454,945 and 6,488,896 to Weigl et al., 6,656,431 toHoll et al., and 6,794,877 to Blomberg et al. Patent application2002/117,517 to Unger et al. is also relevant. Each above-referenceddocument is hereby incorporated by reference, as though set forth hereinin their entireties, for their disclosures of relevant technology andstructure employed in various sensor arrangements.

While considerable progress has been made in sensor technology, a needremains for sensors adapted to interrogate particles that are entrainedin a conductive fluid, which are low in cost, permit samplemanipulation, and/or ensure accurate selection of a sample volume. Itwould be an improvement to provide a sensitive and accurate sensorembodied on a cartridge that is sufficiently low in cost to permit itsdisposal after a single use. It would be another improvement to providesuch a cartridge structured to permit interrogation of a defined samplevolume. Still further improvements would provide verification of samplepresence at one or more desired position in the sensor, and permitestimation of the flow rate of the sample.

BRIEF SUMMARY OF THE INVENTION

This invention provides certain electrically active sensors and methodsof use of those sensors. One operable embodiment of such sensorsincludes a sensor component formed from a plurality of stacked planarthin film layers. Certain of the layers carry one or more electrode todispose a plurality of electrical conductors in a 3-dimensional array inspace. A first portion of a fluid path disposed inside an exemplarysensor passes through at least one layer. A second portion of the fluidpath and a third portion of the fluid path are disposed parallel to, andwithin, the layers, and are disposed on opposite sides of a particleinterrogation zone. Desirably, structure associated with the firstportion is sized to urge particles entrained in a carrier fluid intosubstantially single-file travel through the particle interrogationzone.

Certain preferred embodiments include a first electrode having a surfacegreater than about ⅕ cm² disposed for contact with fluid in the secondportion, and a second electrode having a surface greater than about ⅕cm² disposed for contact with fluid in the third portion of the fluidpath. A sensor may sometimes include a third electrode disposeddownstream from the first electrode, and a fourth electrode disposeddownstream from the third electrode. In such case, the particleinterrogation zone typically comprises a volume disposed between thethird electrode and fourth electrode. Operable sensors may beconstructed having one, two, three, four, or more electrodes.

A sensor may also include flow termination structure disposed downstreamfrom the interrogation zone. Operable flow termination structure isarranged to resist further flow of fluid through the interrogation zonesubsequent to e.g. processing a sample having a known volumetric size.

A sensor may include fluid detection structure arranged to permitnonvisual verification of presence of sample fluid at one or morelocation in the sensor. Sometimes, a sensor may include flow detectionstructure arranged to permit estimation of rate-of-flow of sample fluiddownstream from the interrogation zone. Certain sensors include aparticle filter disposed upstream of the interrogation zone. An operablesuch filter includes openings sized smaller than a cross-section of theinterrogation zone.

A method of use of the apparatus includes: infusing a dose of fluid intoa receiving chamber associated with a sensor; applying a fluid motivesource to the sensor effective to cause fluid from the dose to flowthrough the sensor; applying an electric stimulation signal tostimulated electrodes of the sensor and detecting an electric datasignal received from at least one interrogation electrode associatedwith the interrogation zone; activating a fluid detection portion of thesensor effective to determine arrival of a wave-front of the dose at afirst location disposed downstream of the interrogation zone; andmonitoring the data signal as fluid from the dose continues to flowthrough the interrogation zone. In certain cases, additional fluid flowthrough the interrogation zone is automatically resisted by structure ofthe sensor subsequent to processing a portion of the dose having a knownvolumetric size. The method may also include detecting the wave-front ata second location spaced apart downstream from the first location by aknown volume; and estimating the volumetric flow rate of the dose.Detection of a fluid wave-front at particular locations in the sensormay sometimes be used as triggering events for certain activity, such asstarting and stopping collection of data.

The device may be embodied as a multi-layer microfluidic sensor. In anexemplary such embodiment, a first fluid-flow channel is formed in afirst layer, with the first fluid-flow channel being configured topermit fluid flow in a direction generally parallel to the first layer,and a depth of the channel being less than about 2 mm. A first electrodeof such exemplary embodiment is disposed for contact with fluid in thefirst fluid-flow channel. A second electrode is disposed downstream ofthe first electrode. A second fluid-flow channel passes through a secondlayer, with the second fluid-flow channel being sized to urgesubstantially single-file travel there-through of particles entrained ina carrier fluid. A third electrode is disposed for contact with fluid ina third fluid-flow channel, with the third fluid-flow channel beingformed in a third layer and configured to permit flow of fluid receivedfrom the second fluid-flow channel to continue in a direction generallyparallel to the third layer. Desirably, the first electrode and secondelectrode are carried on a first side of the second layer; and the thirdelectrode is carried on a second side of the second layer. Themulti-layer sensor may include a fourth electrode disposed to cooperatewith the first, second, and third electrodes. Desirably, the firstelectrode and second electrode are carried on a first side of aninterrogation layer, and the third electrode and fourth electrode arecarried on a second side of the interrogation layer.

An operable interrogation layer may include the second layer, only, oradditional layers also. It is within contemplation for certain operableembodiments to provide only a single particle detecting electrodedisposed downstream of the interrogation zone, in which case a stimulussignal may be applied to the electrolytic fluid at a location remotefrom the sensor. It is further within contemplation for certain operableembodiments to provide a particle detecting electrode arrangementincluding an electrode disposed on each side of the interrogation zone.

A multi-layer sensor may also include a first cap layer configured andarranged to provide a boundary surface for the first fluid-flow channel,and a second cap layer configured and arranged to provide a boundarysurface for the third fluid-flow channel. In such case, a first fluidvia passing through the first cap layer is typically configured andarranged for communication with the first fluid-flow channel to permitintroduction of sample fluid into the sensor. Also, a second fluid viapassing through the second layer is typically configured and arranged topermit fluid communication from the third fluid-flow channel to an exitfrom the sensor. It is within contemplation for a layer to be embossedto provide fluid channel structure. In certain sensors, fluid enters athin film portion of the sensor through an entrance port and exits thesensor through an exit port, with the entrance port and exit port beingdisposed on the same side of the thin film portion. A sensor may includefluid detection structure arranged to permit nonvisual verification ofpresence of sample fluid downstream of the third electrode. A sensor mayinclude flow detection structure arranged to permit estimation ofrate-of-flow of sample fluid downstream of the third electrode. A sensormay include holding structure adapted to receive sample fluid effectiveto define a volumetric size of a processed sample. One operable holdingstructure includes a dead-end chamber defining, in harmony with triggerstructure of the sensor, a known volume and being vented through a fluidbarrier effective to resist further flow of fluid through the sensorsubsequent to filling of the chamber.

A device constructed according to certain principles of the instantinvention forms a microfluidic sensor including electrodes adapted tocharacterize particles entrained in a fluid. Such sensor includes aplurality of stacked planar thin film layers cooperatively configured todefine a fluid conduit through the sensor. Preferably, a constrictionportion of the fluid conduit is sized to urge particles intosubstantially single-file travel through a particle interrogation zone.A first pair of electrodes may be disposed upstream of the constrictionportion; and a second pair of electrodes may be disposed downstream ofthe constriction portion. Desirably, the first pair of electrodes iscarried on one side of a first layer of the sensor, and least oneelectrode of the second pair of electrodes is carried on the oppositeside of the first layer. Such sensor may also include a flow terminationstructure configured and arranged to resist further flow of fluidthrough the interrogation zone subsequent to processing a dose of fluidhaving a known volume.

Sometimes, the sensor may include first fluid detection structurearranged to determine arrival of a wave-front of fluid at a firstlocation inside the conduit. Certain sensors may also include secondfluid detection structure arranged to determine arrival of a wave-frontof fluid at a second location disposed downstream from the firstlocation, with the first location and second location being spaced apartby a stretch of conduit having a known volume. Sometimes, the firstlocation is disposed downstream from the interrogation zone. Sometimes,the first location may be disposed downstream from all electrodesoperable on the interrogation zone.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, which illustrate what are currently considered to bethe best modes for carrying out the invention:

FIG. 1 is a cross-section schematic of a multi-layer sensor componentstructured according to certain principles of the instant invention;

FIG. 2 is a top view of an exemplary multi-layer sensor component;

FIG. 3 is an exploded assembly view in perspective from above of themulti-layer sensor component of FIG. 2;

FIG. 4 is an exploded assembly view in perspective from below of themulti-layer sensor component of FIG. 2;

FIG. 5 is a bottom view of the multi-layer sensor component of FIG. 2;

FIG. 6 is a view in perspective from above of a sensor component carriedby a cartridge;

FIG. 7 is an exploded assembly view in perspective from above of thecartridge of FIG. 6;

FIG. 8 is a top view of the cartridge of FIG. 6;

FIG. 9 is a bottom view of the cartridge of FIG. 6;

FIG. 10 is an end view of the cartridge of FIG. 6;

FIG. 11 is a side view of the cartridge of FIG. 6;

FIG. 12 is a top view of a cartridge in position for its installationinto an interrogation platform;

FIG. 13 is a cross-section view, taken through section 13-13 in FIG. 12,and looking in the direction of the arrows;

FIG. 14 is a side view of the assembly of FIG. 12;

FIG. 15 is a cartridge-loading end view of the interrogation platform ofFIG. 12;

FIG. 16 is a schematic of a workable interrogation circuit for use witha sensor such as illustrated in FIG. 6;

FIG. 17 is an X-Y plot of certain interrogation data; and

FIG. 18 is a bar chart depicting certain collected data.

DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENT(S)

Reference will now be made to the drawings in which the various elementsof the illustrated embodiments will be given numerical designations andin which the invention will be discussed so as to enable one skilled inthe art to make and use the invention. It is to be understood that thefollowing description is only exemplary of the principles of the presentinvention, and should not be viewed as narrowing the claims whichfollow.

Currently preferred embodiments of the present invention providelow-cost, disposable, sensors operable to perform analyses of varioussorts on particles that are carried in a fluid. Sensors structuredaccording to certain principles of the instant invention may be usedonce, and discarded. However, it is within contemplation that suchsensors may alternatively be reused a number of times.

Examples of analyses in which embodiments of the invention may be usedto advantage include, without limitation, counting, characterizing, ordetecting members of any cultured cells, and in particular blood cellanalyses such as counting red blood cells (RBCs) and/or white bloodcells (WBCs), complete blood counts (CBCs), CD4/CD8 white blood cellcounting for HIV+ individuals; whole milk analysis; sperm count in semensamples; and generally those analyses involving numerical evaluation orparticle size distribution for a particle-bearing fluid (includingnonbiolgical). Embodiments of the invention may be used to provide rapidand point-of-care testing, including home market blood diagnostic tests.Certain embodiments may be used as an automated laboratory research cellcounter to replace manual hemacytometry. It is within contemplation tocombine the instant invention with additional diagnostic elements, suchas fluorescence, to permit sophisticated cellular analysis and counting(such as CBC with 5-part WBC differential). It is further contemplatedthat embodiments of the invention may be adapted to provide a low-costfluorescence activated cell sorter (FACS).

For convenience in this disclosure, the invention will generally bedescribed with reference to its use as a particle detector. Suchdescription is not intended to limit the scope of the instant inventionin any way. It is recognized that certain embodiments of the inventionmay be used simply to detect passage of particles, e.g. for counting.Other embodiments may be structured to determine particlecharacteristics, such as size, or type, thereby permittingdiscrimination analyses. Furthermore, for convenience, the term “fluid”may be used herein to encompass a fluid mix including a fluid baseformed by one or more diluents and particles of one or more typessuspended or otherwise distributed in that fluid base. Particles areassumed to have a characteristic “size”, which may sometimes be referredto as a diameter, for convenience. Currently preferred embodiments ofthe invention are adapted to interrogate particles found in whole bloodsamples, and this disclosure is structured accordingly. However, such isnot intended to limit, in any way, the application of the invention toother fluids including fluids with particles having larger or smallersizes, as compared to blood cells.

In this disclosure, “single-file travel” is defined different thanliterally according to a dictionary definition. For purpose of thisdisclosure, substantially single-file travel may be defined as anarrangement of particles sufficiently spread apart and sequentiallyorganized as to permit reasonably accurate detection of particles ofinterest. In general, we shoot for single particle detection at leastabout 80% of the time. When two particles are in the interrogation zoneat the same, it is called coincidence, and there are ways tomathematically correct for it. Calibration may be performed usingsolutions having a known particle density (e.g. solutions of latex beadshaving a characteristic size similar to particle(s) of interest). Also,dilution of the particles in a fluid carrier may contribute toorganizing particle travel. As a non-limiting example, it is currentlypreferred to use sensor devices structured to have sizes disclosed inthis document for interrogation of fluid samples having a particledensity of approximately between about 3×10³ to about 3×10⁵ cells/ml,where the particle size is on the order of the size of a red blood cell.

FIG. 1 illustrates certain operational details of a currently preferredsensor component, generally indicated at 100, structured according tocertain principles of the instant invention. As illustrated, sensor 100includes a sandwich of five layers, which are respectively denoted bynumerals 102, 104, 106, 108, and 110, from top-to-bottom. A firstportion 112 of a conduit to carry fluid through the sensor component 100is formed in layer 108. Portion 112 is disposed parallel to, and within,the layers. A second portion 114 of the fluid conduit passes throughlayer 106, and may be characterized as a tunnel. A third portion 116 ofthe fluid conduit is formed in layer 104. Fluid flow through the conduitis indicated by arrows 118 and 118′. Fluid flowing through the first andthird portions flows in a direction generally parallel to the layers,whereas fluid flowing in the second portion flows generallyperpendicular to the layers.

It is within contemplation that two or more of the illustrated layersmay be concatenated, or combined. Rather than carving a channel out of alayer, a channel may be formed in a single layer by machining or etchinga channel into a single layer, or by embossing, or folding the layer toinclude a space due to a local 3-dimensional formation of thesubstantially planar layer. For example, illustrated layers 102 and 104may be combined in such manner. Similarly, illustrated layers 108 and110 may be replaced by a single, concatenated, layer.

With continued reference to FIG. 1, middle layer 106 carries a pluralityof electrodes arranged to dispose a plurality of electrodes in a3-dimensional array in space. Sometimes, such electrodes are arranged topermit their electrical communication with electrical surface connectorsdisposed on a single side of the sandwich, as will be explained furtherbelow. As illustrated, fluid flow indicated by arrows 118 and 118′passes over a pair of electrodes 120, 122, respectively. However, inalternative embodiments within contemplation, one or the other ofelectrodes 120, 122 may not be present. Typically, structure associatedwith flow portion 114 is arranged to urge particles, which are carriedin a fluid medium, into substantially single-file travel through aninterrogation zone associated with one of, or both of, electrodes 120,122. Electrodes 120, 122 may sometimes be made reference to asinterrogation electrodes. In certain applications, an electricalproperty, such as a current, voltage, resistance, or impedance indicatedat V_(A) and V_(B), may be measured between electrodes 120, 122, orbetween one of, or both of, such electrodes and a reference.

Certain sensor embodiments employ a stimulation signal based upondriving a desired current through an electrolytic fluid conductor. Insuch case, it can be advantageous to make certain fluid flow channelportions approximately as wide as possible, while still achievingcomplete wet-out of the stimulated electrodes. Such channel width ishelpful because it allows for larger surface area of the stimulatedelectrodes, and lowers total circuit impedance and improves signal tonoise ratios. Exemplary embodiments used to interrogate blood samplesinclude channel portions that are about 0.10″ wide and about 0.003″ highin the vicinity of the stimulated electrodes.

One design consideration concerns wettability of the electrodes. At someaspect ratio of channel height to width, the electrodes MAY not fullywet in some areas, leading to unstable electrical signals and increasednoise. To a certain point, higher channels help reduce impedance andimprove wettability. Desirably, especially in the case of interrogationelectrodes, side-to-side wetting essentially occurs by the time thefluid front reaches the second end of the electrode along the channelaxis. Of course, wetting agents may also be added to a fluid sample, toachieve additional wetting capability.

Still with reference to FIG. 1, note that electrodes 120 and 122 areillustrated in an arrangement that promotes complete wet-out of eachrespective electrode independent of fluid flow through the tunnelforming flow portion 114. That is, in certain preferred embodiments, theentire length of an electrode is disposed either upstream or downstreamof the tunnel forming flow portion 114. In such case, the “length” ofthe electrode is defined with respect to an axis of flow along a portionof the conduit in which the electrode resides. The result of such anarrangement is that the electrode is at least substantially fully wettedindependent of tunnel flow, and will therefore provide a stable,repeatable, and high-fidelity signal with reduced noise. In contrast, anelectrode having a tunnel passing through itself may provide an unstablesignal as the wetted area changes over time. Also, one or more bubblemay be trapped in a dead-end, or eddy-area disposed near the tunnel(essentially avoiding downstream fluid flow), thereby variably reducingthe wetted surface area of a tunnel-penetrated electrode, andpotentially introducing undesired noise in a data signal.

In general, disposing the electrodes 120 and 122 closer to the tunnelportion 114 is better (e.g., gives lower solution impedancecontribution), but the system would also work with such electrodes beingdisposed fairly far away. Similarly, a stimulation signal (such aselectrical current) could be delivered using alternatively structuredelectrodes, even such as a wire placed in the fluid channel at somedistance from the interrogation zone. The current may be delivered fromfairly far away, but the trade off is that at some distance, theelectrically restrictive nature of the extended channel will begin todeteriorate the signal to noise ratios (as total cell sensing zoneimpedance increases).

With continued reference to FIG. 1, electrode 124 is disposed forcontact with fluid in conduit flow portion 112. Electrode 126 isdisposed for contact with fluid in flow portion 116. It is currentlypreferred for electrodes 124, 126 to also be carried on a surface ofinterrogation layer 106, although other configurations are alsoworkable. Note that an interrogation layer, such as an alternative toillustrated single layer 106, may be made up from a plurality ofsub-component layers. In general, electrodes 124, 126 are disposed onopposite sides of the interrogation zone, and may sometimes be madereference to as stimulated electrodes. In certain applications, a signalgenerator 128 is placed into electrical communication with electrodes124 and 126 to input a known stimulus to the sensor 100. However, it iswithin contemplation for one or both of electrodes 124, 126 to not bepresent in alternative operable sensors structured according to certainprinciples of the instant invention. In alternative configurations, anyelectrode in the sensor 100 may be used as either a stimulated electrodeor interrogation electrode.

One currently preferred sensor component, generally indicated at 130,will now be described with reference to FIGS. 2-5. Sensor 130 includesfive thin film layers stacked to form a thin film sandwich, similar tothe embodiment depicted in FIG. 1. FIG. 2 is a top view of sensorcomponent 130, and shows how the top cap layer 102 and top channel layer(e.g. 104, FIG. 3) form a window arranged to permit access to a portionof interrogation layer 106. In the illustrated embodiment, the exposedportion includes an edge of layer 106. The exposed surface of the edgeof interrogation layer 106 carries a plurality of conductors (134through 146, respectively) that are configured to form an electricalinterface to interrogation circuitry. That is, a portion of each ofconductors 134-146 is desirably exposed to form a plurality of surfaceconnectors of an electrically communicating interface. One operable suchinterface may be formed in harmony with a commercially availablemulti-pin electrical connector, such as part No. SIB-110-02-F-S-LC,available from Samtec having a place of business located in New Albany,Ind. Other workable connector structure includes touch-down probes, andother electrically-conductive, contact-forming probes known in the art.

Also shown in FIG. 2 are alignment holes 148 and 150, respectively.Because top layer 102 is illustrated as being transparent (although suchis not required for practice of the invention), electrode 126 disposedin channel portion 116 is visible. Similarly, fluid via 152 may be seen.As will be detailed further below, via 152 passes through interrogationlayer 106, and permits fluid flow downwardly through the thickness ofthe sensor component 130. Electrode 122 is also visible, disposed inassociation with the interrogation zone, generally indicated at 154.

With particular reference now to FIGS. 3 and 4, a pair of fluid viaspass through bottom cap layer 110. Via 156 is a fluid entrance via,through which sample fluid enters the sensor component 130 for continuedflow through channel portion 112, as indicated by fluid flow directionarrow 158. Channel portion 112 is disposed in layer 108 and introducesfluid into the interrogation zone 154 (or tunnel-like channel portion114 in FIG. 1). Downstream from the interrogation zone 154, fluid flowsthrough channel portion 116 as indicated by fluid flow direction arrow158′. Channel portion 116 is disposed in layer 104 and communicates tovia 152 passing through layer 106. Fluid via 152 communicates fluid intochannel portion 160 disposed in layer 108. Fluid via 162 is a fluid exitvia, through which fluid flowing through channel portion 160 may leavethe sensor component 130. A direction of fluid flow in channel 160 isindicated by arrow 158″.

As best seen with reference to FIGS. 4 and 5, certain embodiments of asensor component 130 may include one or more additional and optionalelectrodes. Layers 102 and 104 are illustrated as being transparent,although such is not required for practice of the invention. Asillustrated, electrodes 164, 166, and 168 are disposed on layer 106 andare arranged for contact with fluid carried in channel portion 160.Electrode 164 is in electrical communication with conductor 146;electrode 166 is in electrical communication with conductor 142; andelectrode 168 is in electrical communication with conductor 140. Suchoptional electrodes may be used, for examples, to verify the presence ofsample fluid at one or more known locations in sensor 130, to estimatethe rate of fluid flow through the sensor, and/or as start or stoptriggers for an activity such as data acquisition.

It should be noted that certain electrodes carried by sensor 130 (e.g.120, 124, 164-168), are in electrical communication with theirrespective conductor that is disposed on an opposite side of layer 106by way of a conductive path disposed through a respective electrical via170 (see FIG. 3). Such conductive path is conveniently formed during alaminating or metallizing step during manufacture of the sensorcomponent. In any case, it should be appreciated that a complex patternof electrodes can be disposed to interrogate fluid in 3-dimensionalspace, even in the illustrated case where the electrodes are carried bya single metallized layer.

The conductive elements forming conductors (e.g. 134-146) and/orelectrodes (e.g. 120-126) must simply conduct electricity, and caninclude one or more metal, such as Copper, Silver, Platinum, Iridium,Chromium, and Gold, or alloys, or multiple layers of metals or alloys.The vias 170 permit conduction of electricity from top to bottom throughspacer layer 106, and enable surface conductors to be disposed on onlyone side of the spacer layer, for convenient interface with acommercially available electrode interface (i.e. connector). Of course,it is realized that certain interface probe-electrodes of aninterrogation platform may be structured to avoid vias on the sensor,e.g. that surface electrodes can be provided on both sides of the spacerlayer, in alternative sensor constructions.

An electrical property at an electrode may be monitored to determinearrival of fluid at that electrode. For example, the impedance measuredat an electrode undergoes a significant change in value as thewave-front, or the leading edge, an electrolyte fluid passes over theelectrode. In one currently preferred use of the sensor component 130(see FIG. 4), a stimulus electric signal (such as a 1 kHz square wave)is applied to electrode 164. A sudden change in the impedance valuesmeasured at electrodes 166 and 168 indicates the successive arrivals ofthe wave-front of the sample fluid at each respective electrode. In theillustrated embodiment 130, first verification of fluid at electrode 166ensures that sample fluid is in place for interrogation, and a test runcan begin. Feedback from electrode 166 may therefore serve as a firsttrigger to begin interrogation of the fluid sample.

A change in impedance at electrode 168 indicates the wave-front hasreached that electrode as well. A time differential between theimpedance changes at electrodes 166 and 168 can be used, in harmony witha known volume there-between, to estimate a fluid flow rate through thesensor component 130. The volume between electrodes 166 and 168 may becalculated by integrating the function of the cross-section area ofchannel portion 160 along the length L1 of such channel portion disposedbetween those electrodes. It is currently preferred to simplify suchcalculation by holding the cross-section of channel portion 160 constantbetween electrodes.

Electrodes, such as 166 and 168, may be disposed as first and secondtriggers operable to indicate respective start and stop signals basedupon detection of a fluid boundary. The first and second triggers can belocated to have locations of effective operation that are disposedspaced apart by a lumen defining a known volume. Such triggers may beused, for non-limiting example, to start and stop data acquisition for asample having a known volume. It is preferred for cooperating triggerelectrodes to have substantially the same conformation (e.g. wetted areaand axial length), to promote consistent electrical response of eachsubsequent downstream trigger. Sometimes, the channel may be narrowed inthe vicinity of an electrode to reduce possible variations in the shapeof the fluid front as it makes contact with the electrode.

A sensor 130 may be formed from a plurality of stacked and bonded layersof thin film, such as a polymer film. In an exemplary sensor component130 used in connection with interrogation of blood cells, it iscurrently preferred to form top and bottom layers 102 and 110 fromPolyamide or Mylar film. A workable range in thickness for Polyamidelayers is believed to be about 0.1 micron to about 500 microns. Acurrently preferred Polyamide layer 102, 110 is about 52 microns inthickness. It is further within contemplation that a pair of top and/orbottom layers can be formed from a single layer including fluid channelstructure formed e.g. by etching, molding, or hot embossing.

It is currently preferred to make the spacer layer 106 from Polyamidealso. However, alternative materials, such as Polyester film or Kapton,which is less expensive, are also workable. A film thickness of about 52microns for spacer layer 106 has been found to be workable in a sensorused to interrogate blood cells. Desirably, the thickness of the spacerlayer is approximately on the order of the particle size of the dominantparticle to be interrogated. A workable range is currently believed tobe within about 1 particle size, to about 15 times particle size, or so.

Vias 170 are typically formed in the layer 106 prior to dual-sideddeposition of the conductive elements onto such layer, althoughalternative manufacturing techniques are workable. Alignment apertures148, 150 and via 152 may be formed at the same time as vias 170, orsubsequent to the metallizing step. Such void elements, and channelportions, may be formed by cutting through the respective layer with alaser, water jet, die stamping, drilling, or by some other machiningtechnique. Deposition of conductive film elements to layer 106 may beeffected using well-known metal-deposition techniques, includinglamination. Metal sheets may be laminated to a polymer layer using thinadhesive. Double clad sheets formed in such manner are commerciallyavailable, and can be patterned as desired to form electrodes. It isbelieved that workable sensors can be made having test electrodes thatare 0.5 microns in thickness, or perhaps even less. Electrodes for usein currently preferred blood cell sensors may be up to about 36 micronsin thickness. Sometimes, a pair of metals, such as Cu or Cr and Au maybe deposited in the current process. The Cu or Cr layer may be thin,typically goes on first, and acts as a bonding layer between the polymerfilm and the Au. It is currently preferred to configure the electrodesand conductive elements by wet etching subsequent to deposition of theelectrically conductive material.

Impedance at the electrode/electrolyte interface is proportional towetted electrode surface. Electrodes may be configured having a desireduseful size of surface area disposed for contact with fluid in achannel. It is currently preferred to apply a stimulation signal tostimulated electrodes to cause at least about 0.1 mA RMS current flowthrough the interrogation zone. The currently preferred signal is at 100kHz, although signals at lower frequency or higher frequencies, such as200 k Hz, or more, are operable. The surface area of the stimulatedelectrodes are sized to accommodate a desired current flow and signalfrequency. It is currently believed that electrodes should be sized tohave a current density of less than about 5 mA/cm².

In one embodiment of sensor 130 adapted to impart a constant 1 mA RMScurrent stimulation at about 100 kHz, interrogation electrodes 120, 122have a wetted surface area of about 0.036 cm², and stimulated electrodes124, 126 have a wetted surface area of about 0.45 cm². In such case, itis thought that the stimulated electrodes 124, 126 could be reduced insize to about ⅕ cm², or less, without suffering a lack of performancedue to degradation of the electrode during such stimulation.

The channel portion 114 is typically laser drilled through layer 106(and any electrodes carried thereon that are also disposed in the fluidpath). A diameter of 35 microns for channel 114 is currently preferredto urge blood cells toward single-file travel through the interrogationzone 154. Other cross-section shapes, other than circular, can also beformed during construction of channel 114. Naturally, the characteristicsize of the orifice formed by drilling channel 114 will be dependentupon the characteristic size of the particles to be characterized orinterrogated. Counter-boring can be performed on thicker layers toreduce the “effective thickness” of the sensing zone.

Alignment holes 148, 150 passing through each layer may be used to alignthe various layers using guide pins during assembly of the plurality oflayers. A double-sided adhesive polymer film is currently preferred as amaterial of composition for combination bonding-channel layers 104 and108. Layers 104 and 108 in a currently preferred sensor 130 are madefrom double-sided Polyamide (PET) tape having a thickness of about0.0032 inches. Alternatively, a plain film layer may be laminated to anadjacent plain layer using heat and pressure, or adhesively bonded usingan interposed adhesive, such as acrylic or silicone adhesive.

A currently preferred sensor structured according to certain principlesof the instant invention is generally indicated at 180 in FIG. 6, andmay sometimes be characterized as a cartridge. Cartridge 180 includes abase 182 on which to hold a sensor component, such as some sort of thinfilm sensor 183. A workable base may be formed by injection molding aplastic, or plastic-like, material. It is preferred to configure base182 having a small size to reduce a required volume of constituentmaterial, but still form a sensor 180 that is sufficiently large tofacilitate its handling and manipulation under control of a human hand.

With reference now to FIG. 7, base element 182 includes alignment holes184, 186, and 188, which also extend to pass through channel layer 190,cap layer 192, and adhesive layer 194. Holes 184 and 186 are configuredand arranged to cooperate with alignment holes 148 and 150,respectively, to permit component alignment using pin elements of acommon fixture during assembly of the sensor 180. Base element 182 alsoholds sample receiving chamber 196, and processed fluid chamber 198.Vent aperture 200 is in fluid communication through base 182 with ventconnection access port 202. Similarly, vacuum aperture 204 is in fluidcommunication through base 182 with vacuum connector access port 206.

Channel layer 190 may be formed from a thin film of polymer film,similar to layers 104, 108 of the sensor 130. Preferably, layer 190 ismade from a two-sided adhesive tape, such as Polyamide tape. Layer 190includes cut-out area shaped to form additional void elements, includingchannel 208, a portion of which augments a volume provided by chamber196 in which to receive a fluid sample. Transverse portion 210 ofchannel 208 communicates to vent aperture 200, effective to permitescape of air from chamber 196 during infusion of a sample forinterrogation.

Continuing to refer to FIG. 7, aperture 212 extends as a fluid-flowchannel or via through layers 190, 192 and 194 effective to introducefluid received from chamber 196 into sensor component 183. An optionallyenlarged portion of channel 212 permits fluid to spread out over asufficiently large filter area prior to passing through optional filterelement 214 and entering sensor element 183. It typically is desirableto include a filter element 214 to resist entrance into the interior ofsensor component 183 of clots or debris that might plug channel portion114. A preferred filter element 214 resists passage of particles largerthan those approaching the characteristic size of the interrogation zone154. A workable filter 214 includes a Nylon Net Filter NY30 availablefrom Millipore Cat: NY3004700, which has filtering pores that are about30 microns in size. Desirably, the filter essentially consists ofopenings having a characteristic size that is smaller than acharacteristic size of a minimum cross-section of the interrogationzone.

Still with reference to FIG. 7, layer 190 also includes vacuum channel216, which communicates at end 218 with vacuum aperture 204. As will bediscussed in more detail below, fluid may be transported through certainconduits of sensor 180 using a vacuum source that may be connected toport 206.

In certain preferred embodiments, a barrier element 220 is disposed inassociation with aperture 222 passing through layer 190. A workablebarrier element 220 permits escape of air from chamber 198, but resistsescape of fluid from such chamber. A preferred barrier 220 includes aPTFE gasket, such as a 0.2 micron pore size Fluoropore, FGLP, which canbe purchased from Millipore Cat. No. FGLP01300. Gasket 220 isillustrated in FIG. 7 as being installed in a preferred blockingposition on the bottom of layer 190, but may be disposed in a blockingposition on either side of layer 190. Barrier 220 is an exemplaryembodiment of flow termination structure disposed downstream from theinterrogation zone and arranged to resist flow of fluid beyond aboundary associated with the microfluidic sensor. Other operable flowtermination structure includes porous materials that turn to gels whenwet; hydrophobic porous membranes or plugs; and very small laser drilledholes in films (e.g. <10 microns).

Continuing to refer to FIG. 7, an exemplary layer 192 may be made frompolymer film, and functions as a cap layer, similar to layer 110 ofsensor component 183. An embossed portion 224 is formed in layer 192 tocreate a simple channel structure through which air can communicatebetween end 226 of vacuum channel 216 and aperture 222. The vacuum-sidefluid conduit communicating between port 206 and a sensor exit (such asexit via 162 of sensor 130), is completed by way of aperture 228, whichforms a fluid conduit or via extending from end 230 of chamber 198,through layers 190, 192, and 194, for communication with a fluid exitvia of an installed thin film sensor component 183.

In use of the device, a micro-pipette tip may be inserted forfluid-tight reception into sample-receiving aperture or port 230. A rawfluid sample can then be infused from the micro-pipette into chamber196, while air is permitted to escape through channel 210 and vent port202. The size for a raw fluid sample for characterization of blood cellsin a representative device is 50 μl, although the sensor conduits andchambers may be sized to accommodate samples having an alternativedesired size. Vent port 202 is then occluded, either manually or usingan automated structure. A vacuum source is then applied to port 206 topromote fluid flow from holding chamber 196, through channel 208,aperture 212, optional filter 214, and into a fluid entrance of thesensor component.

After flowing through the sensor component, fluid is drawn throughaperture 228 and into holding chamber 198. Once chamber 198 is filled,fluid is barred from further flow by barrier element 220, which is oneexample of operable flow termination structure that resists additionalflow. The volume of fluid encompassed by chamber 198 can help todetermine a known volume for processed fluid. In the representativedevice, the processed fluid volume, defined by chamber 198 incombination with a small upstream volume contained in conduit structurestretching to a fluid-front presence verification structure, such aselectrode 166 (see FIG. 4), is 25 μl.

Additional details of construction of an exemplary cartridge 180 areillustrated in FIGS. 8-11. Notably, ramp structure 232, best seen inFIGS. 9 and 10, can be helpful to assist in coupling the cartridge withcertain interrogation platforms. Other structure associated with thebase 182, such as alignment holes 186 and 188, may also be employed toassist in coupling a cartridge with an interrogation platform.

An interrogation platform desirably provides three functions; 1)electrical continuity to the sensor, 2) fluid-flow control, and 3)alignment. A workable, and currently preferred interrogation platform isindicated generally at 240 in FIGS. 12-15.

A cartridge 242 is illustrated in position for its insertion inregistration with socket 244 (see FIG. 13). As the cartridge is insertedinto the socket 244, ramps 232 on the bottom side of the cartridge bodypress the two alignment pins 246 down. The cartridge 242 then comes intocontact with the vent and vacuum connectors, 248 and 250, respectively.

In the illustrated platform 240, the vent connector 248 and vacuumconnector 250 are made from silicone rubber tubing. The rubber tubesmate with respective connection ports (e.g. 202 and 206, see FIG. 10) toform an airtight seal. The silicone rubber tubing is supported on theinside by a smaller, more ridge piece of tubing. The rigid, internaltubing imparts the required mechanical stability while the soft,flexible rubber tubing conforms to make an airtight seal. This airtightseal is actually made because the rubber tube extends sufficiently tocontact the bottom of the cartridge mating hole before the cartridge isfully seated. When the cartridge is inserted slightly further into theinterrogation platform, the rubber tube is forced to expand radiallyoutward, thereby making an airtight seal against its receiving socket(e.g. 202 or 206).

When seated in socket 244, the electrical contact pads (on the top ofthe cartridge and generally indicated at 252 in FIG. 12) contact biasedpins 254 of the electrical connector 256. The electrical connector 256places the sensor 242 in electrical communication with test circuitry257 that is typically carried by an interrogation platform and isadapted to interrogate particles passing through the sensor.

When the cartridge 242 is fully inserted, the alignment pins 246 seatinside the alignment holes 184, 186 in the bottom of the cartridge viaforce imparted by springs 258. The cartridge is now fully engaged,aligned, and ready for testing. To remove a cartridge, the release latch260 is pressed downward, thereby retracting the alignment pins 246 fromthe cartridge body as the latch rotates about pivot axle 262. Thecartridge can then be easily pulled out of the interrogation platform.

The interrogation platform may include circuitry that may be carried onprinted circuit board 264, or otherwise arranged to communicate with thesensor. A plurality of different test circuits may be provided by simplyexchanging the circuit board to one having the desired configuration.Such circuitry may include structure arranged to apply a firsttime-varying stimulus signal to stimulated electrodes. A currentlypreferred first stimulus signal is a constant current source, although aconstant voltage source is also workable. A preferred first stimulussignal is about 100 kHZ 1 mA rms. A second stimulus signal may beprovided and coupled to electrodes adapted to detect presence of a fluidwave-front. A preferred second signal is a 1 k Hz square wave input to afirst electrode and permitting measurement of an electric property byusing at least one other electrode. Impedance or voltage may beevaluated at or between measurement electrodes. Sometimes, adifferential may be measured between electrodes. Other times, ground maybe enforced at one electrode, and an electrical property measured at theother electrode. It is within contemplation for one electrode to beeliminated entirely, and use a ground reference.

FIG. 16 illustrates one workable interrogation circuitry adapted tointerrogate a sensor 130. In the cell interrogation loop, a signalgenerator 268 is applied between conductors 134 and 144. It has beendetermined that electrical ground may be enforced at one of suchconductors. In current sensors adapted to interrogate blood cells, it ispreferred to apply a constant RMS current signal of about 1 mA at about100 kHz. Values for voltages V_(A) and V_(B) are measured at conductors136 and 138, respectively for calculation of a differential voltageacross the cell interrogation zone. It has been determined that anelectrical ground may be enforced at one such conductor, and the voltagedirectly measured at the other conductor may be used in place of a truedifferential voltage. It is also possible to allow one electrode to“float” (i.e. not be connected to anything) and measure the voltage fromthe other electrode. In the fluid detection loop, a signal generator 270is applied between conductor 146 and each of conductors 140 and 142. Theimpedance or voltage signal V_(S1) and V_(S2) are measured to determinethe presence of the fluid wave-front. A sudden drop in the measuredimpedance indicates presence of the wave-front of electrolytic fluid. Aworkable signal includes a square-wave at about 1 kHz at about 3 volts.FIGS. 17 and 18 present data that may be collected using the disclosedsensors.

In a method of using the device to count cells in a blood sample, 50micro-liters of fluid are added to the sensor via the pipette-tip holewhich is sized to form an air tight fit with the pipette tip. As thesample enters the sample storage channel, air displaced by the fluidexits the cartridge through a vent port that connects to theinterrogation platform. The sample can be added to the cartridge beforeor after it has been connected to the interrogation platform. Once thesample is in the cartridge, and the cartridge is installed in aninterrogation platform, the user starts the test by activating one ormore “start” control of the system. The “start” causes a valve connectedto the vent port to close, thereby not allowing the sample to flow intothe vent port. The “start” also opens the vacuum valve to start pullingthe fluid sample into the sensor. Because the vent is sealed, fluid isdrawn from the sample storage chamber and though the thin film sensorcomponent. A “start” may also initiate a stimulus (e.g. 1 kHz) to thesample detection electrodes embedded in the thin film sensor component.Once the fluid is through the sensing orifice and has wet the stimulusand measurement electrodes, it flows over a pair of sample detectionelectrodes. As the fluid wave-front reaches the detection position atthe second electrode, a large drop in electric impedance is detected andthe constant current source is activated (e.g. 100 kHz @1 mA). Adifferential voltage is measured across the interrogation zone (4electrode configuration, currently preferred) and used to determine cellsize (and/or count) subsequent to the time of wave-front detection.Fluid continues to flow until it reaches the end of the “dead-end”channel and no more cells are detected. The volume that is processed ina test run is determined by the volume accommodated downstream of thewave-front detection location, and is 25 micro-liters in a preferreddevice. The method may also include monitoring one or more additionalsample detection electrode placed further down the channel, i.e. todetermine the approximate flow rate during, or prior to starting, thecell counting.

While the invention has been described in particular with reference tocertain illustrated embodiments, such is not intended to limit the scopeof the invention. The present invention may be embodied in otherspecific forms without departing from its spirit or essentialcharacteristics. The described embodiments are to be considered in allrespects only as illustrative and not restrictive. The scope of theinvention is, therefore, indicated by the appended claims rather than bythe foregoing description. All changes which come within the meaning andrange of equivalency of the claims are to be embraced within theirscope.

1. A microfluidic sensor, comprising: a plurality of stacked thin filmsubstantially planar layers, certain of said layers carrying one or moreelectrode to dispose a plurality of electrodes in a 3-dimensional arrayin space; and a fluid path, disposed inside said sensor, comprising: afirst portion disposed parallel to, and within, said layers; a secondportion provided by a tunnel passing through at least one layer, aconstriction in said tunnel being sized to urge particles entrained in acarrier fluid into substantially single-file travel through a particleinterrogation zone; and a third portion disposed parallel to, andwithin, said layers, said first portion and said third portion beingdisposed on opposite sides of said particle interrogation zone.
 2. Thesensor according to claim 1, wherein: an interrogation layer of saidsensor comprises said at least one layer; and at least one electrode iscarried on each of top and bottom surfaces of said interrogation layer.3. The sensor according to claim 2, further comprising: a plurality ofelectrical surface connectors individually disposed in electricalcommunication with selected ones of each of said electrodes; wherein:all of said electrical surface connectors are disposed on a single sideof said interrogation layer.
 4. The sensor according to claim 1, furthercomprising: a first trigger and a second trigger operable to indicaterespective start and stop signals based upon detection of a fluidboundary, said first trigger and said second trigger having respectivelocations of effective operation that are disposed spaced apart by alumen defining a known volume.
 5. The sensor according to claim 4,wherein: said first trigger comprises: a first electrode disposed tocontact fluid flowing through said sensor; and a second electrodedisposed to contact fluid flowing through said sensor, wherein: saidfirst trigger is structured to permit detecting impedance between saidfirst electrode and said second electrode.
 6. The sensor according toclaim 5, wherein: said second trigger comprises: said first electrode;and a third electrode disposed to contact fluid flowing through saidsensor, wherein: said second trigger is structured to permit detectingimpedance between said first electrode and said third electrode.
 7. Thesensor according to claim 1, further comprising: a first stimulatedelectrode disposed for contact with fluid in said first portion; asecond stimulated electrode disposed for contact with fluid in saidthird portion; and a first interrogation electrode disposed for contactwith fluid between said first stimulated electrode and said secondstimulated electrode.
 8. The sensor according to claim 7, wherein: saidfirst stimulated electrode and said second stimulated electrode eachhave a surface area, disposed for contact with fluid in said fluid path,greater than about 1/10 cm².
 9. The sensor according to claim 7, furthercomprising: a second interrogation electrode disposed between said firststimulated electrode and said second stimulated electrode.
 10. Thesensor according to claim 7, wherein: the entire length of said firstinterrogation electrode, measured along an axis of said first portion,is disposed upstream of said tunnel.
 11. The sensor according to claim9, wherein: the entire length of said second interrogation electrode,measured along an axis of said third portion, is disposed downstream ofsaid tunnel.
 12. The sensor according to claim 7, wherein: a secondinterrogation electrode is disposed between said first stimulatedelectrode and said second stimulated electrode. the entire length ofsaid first interrogation electrode, measured along an axis of said firstportion, is disposed upstream of said tunnel; and the entire length ofsaid second interrogation electrode, measured along an axis of saidthird portion, is disposed downstream of said tunnel.
 13. The sensoraccording to claim 7, wherein: the entire length of said firstinterrogation electrode, measured along an axis of said third portion,is disposed downstream of said tunnel.
 14. The sensor according to claim1, further comprising: a particle filter disposed upstream of saidinterrogation zone, said particle filter consisting essentially ofopenings having a characteristic size that is smaller than acharacteristic size of a minimum cross-section of said interrogationzone.
 15. The sensor according to claim 1, further comprising: flowtermination structure disposed downstream from said interrogation zone,said flow termination structure being arranged to resist flow of fluidbeyond a boundary associated with said microfluidic sensor.
 16. Thesensor according to claim 1, further comprising: holding structureadapted to receive a quantity of sample fluid effective to define avolumetric size of a processed sample, said holding structure comprisinga dead-end chamber defining, in harmony with trigger structure of saidsensor, a known volume and being vented through a fluid barriereffective to resist further flow of fluid through said sensor subsequentto filling of said chamber.
 17. A multi-layer microfluidic sensor,comprising: a first fluid-flow channel formed in a first layer, saidfirst fluid-flow channel being configured to permit fluid flow in adirection generally parallel to said first layer; a first electrodedisposed for contact with fluid in said first fluid-flow channel; asecond electrode disposed downstream of said first electrode, said firstelectrode and said second electrode being carried on a first side of aninterrogation layer; a second fluid-flow channel passing through saidinterrogation layer, said second fluid-flow channel being sized to urgesingle-file travel therethrough of particles entrained in a carrierfluid; and a third electrode, disposed for contact with fluid in a thirdfluid-flow channel and carried on a second side of said interrogationlayer, said third fluid-flow channel being formed in a layer andconfigured to permit flow of fluid received from said second fluid-flowchannel to continue in a direction generally parallel to a layer. 18.The sensor according to claim 17, further comprising: flow detectionstructure arranged to permit estimation of rate-of-flow of sample fluiddownstream from said interrogation zone.
 19. The sensor according toclaim 17, further comprising: an entrance port communicating to saidfirst fluid-flow channel; and an exit port communicating from said thirdfluid-flow channel, said entrance port and said exit port being disposedon the same side of said sensor.
 20. The sensor according to claim 17,further comprising: a first trigger and a second trigger operable toindicate respective start and stop signals based upon detection of afluid boundary, said first trigger and said second trigger havingrespective locations of effective operation that are disposed spacedapart by a lumen defining a known volume.